Identification of two SET domain proteins required for methylation of lysine residues in yeast ribosomal protein Rpl42ab.
We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.
Webb
Kristofor J
KJ
Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, California 90095, USA.
Laganowsky
Arthur
A
Whitelegge
Julian P
JP
Clarke
Steven G
SG
D003513:Cycloheximide / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide; D008780:Methyltransferases; D008239:Lysine